My long-term scientific goal is to understand the molecular mechanisms that specify retina cell number. Using the compound eye of Drosophila as an experimental model, my laboratory has discovered the Hippo pathway as a central mechanism underlying this process. The core of the Hippo pathway comprises a kinase cascade in which the Ste20 kinase Hippo (Hpo) phosphorylates and activates the NDR family kinase Warts (Wts). Wts, in turn, phosphorylates and inactivates the oncoprotein Yorkie (Yki) by excluding it from the nucleus, where it normally functions as a coactivator for the DNA-binding transcription factor Scalloped (Sd). Our research further established a critical role for the Hippo pathway in controlling organ size in mammals, underscoring the importance of Drosophila as a powerful model to discover universal developmental mechanisms. Despite recent progress, our understanding of the composition, mechanism and regulation of Hippo signaling remains incomplete. Much of our recent efforts have focused on discovering the missing components of the Hippo pathway, with the ultimate goal of defining a complete Hippo signaling network. In the current grant period, we have filled several key gaps in our understanding of the Hippo pathway, including a functional link between Hippo signaling and the innate immunity receptor Toll, spectrin as an upstream regulator of Hippo signaling by modulating actomyosin, autoinhibition of Hpo activity mediated by the STRIPAK phosphatase complex, a Hpo-like kinase that functions redundantly with Hpo, a histone methyltransferase complex recruited by Yki to activate the transcription of Hippo target genes, and a zinc finger transcriptional repressor recruited by Sd to repress the transcription of Hippo target genes. We further contributed to the Hippo research community by developing a set of fly stocks that can be used to unequivocally validate any Hippo pathway regulators through rigorous genetic epistasis test. In the next grant period, we will further elucidate the molecular underpinnings of the Hippo pathway through the following specific aims. First, we will dissect the molecular and cellular mechanisms by which spectrin and actomyosin cytoskeletons regulate Hippo signaling in developing tissues. Second, we will identify upstream tumor suppressors that regulate Hippo signaling by antagonizing the activity of the STRIPAK phosphatase complex. These studies will allow us to define how the STRIPAK phosphatase complex functions as a central hub that integrates diverse upstream inputs into the Hippo pathway. Lastly, we will characterize novel regulators of Hippo signaling isolated from a sensitized screen for mutations that enhance a partial loss-of-Hippo phenotype in the eye. This unbiased approach will shed light on previously unforeseen regulators/mechanisms underlying the Hippo pathway. Besides revealing fundamental mechanisms of eye development, the proposed studies will have general implications for the development of other tissues.